Methods and reagents for pyranosone production

ABSTRACT

Methods for producing substantially pure pyranosone from pyranose, especially glucosone from glucose. A pyranose-2-oxidase enzyme which is free of substantially all glucosone-utilizing enzyme contaminants which are measurably active at a selected pH between about 4.4 and 7.0 is reacted in solution with a pyranose and oxygen to produce the corresponding pyranosone. Hydrogen peroxide co-produced in the reaction is maintained at a concentration below about 10 -3  M, by substantially pure catalase. The reaction is carried out at the selected pH. The methods and reagents are also applicable to producing xylosone from xylose, 5-ketofructose from sorbose, and mixtures of 2-ketogluconic and isoascorbic acid from delta-gluconolactone.

BACKGROUND AND SUMMARY

The present invention relates to methods and reagents for obtainingsubstantially pure products by enzymatic oxidation of the hydroxyl groupat specific positions on certain pyranoses and structurally relatedcompounds. In particular, the invention concerns improved processes andreagents for making D-glucosone from D-glucose, D-xylosone fromD-xylose, 5-keto-D-fructose from L-sorbose, or a mixture of2-keto-D-gluconic acid and D-isoascorbic acid fromdelta-D-gluconolactone by reacting D-glucose, D-xylose, L-sorbose ordelta-D-gluconolactone, respectively, with oxygen in aqueous solution ata pH between about 4 and 7 in the presence of a pyranose-2-oxidaseenzyme. The invention also concerns pyranose-2-oxidase enzymes and fungiwhich are sources thereof. Finally, the invention pertains topyranosone-utilizing enzymes often found in pyranose-2-oxidase enzymepreparations obtained from certain fungal sources.

As used herein:

Glucose means D-glucose.

Glucosone means D-glucosone, also known as D-arabino-2-hexosulose.

Fructose means D-fructose.

Xylose meand D-xylose. Xylosone means D-xylosone, also known asD-threo-2-pentosulose.

Xyulose means D-xyulose, also known as D-threo-2-pentulose.

Sorbose means L-sorbose.

5-ketofructose means 5-keto-D-fructose, also known asD-threo-2,5-hexodiulose.

Delta-gluconolactone means delta D-gluconolactone, also known asD-gluconic acid delta-lactone.

2-ketogluconic acid means 2-keto-D-gluconic acid.

Isoascorbic acid means D-isoascorbic acid, also known as D-araboascorbicacid.

Pyranose means glucose, xylose, sorbose or delta-gluconolactone.

Pyranosone refers to glucosone or xylosone.

NRRL means the culture depository of the United States Department ofAgriculture Northern Regional Research Laboratory in Peoria, Ill.,U.S.A. All NRRL deposits referred to herein were made under the terms ofthe Budapest Treaty on the International recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and Regulationspromulgated thereunder.

ATCC refers to the American Type Culture Collection culture depositorylocated in Rockville, Md., U.S.A.

CMCC refers to the private culture collection of Cetus Corporation, 1400Fifty-Third street, Emeryville, Calif., U.S.A.

There is commercial interest in producing substantially pure pyranosonesby enzymatic conversion of corresponding pyranoses. In part, thisinterest has grown out of recent advances in fructose production inwhich glucosone is made as an intermediate. U.S. Pat. No. 4,246,347,assigned to the assignee of the present application and incorporatedherein by reference, discloses a fructose-production method comprisingenzymatic conversion of glucose to glucosone, followed by catalytichydrogenation of glucosone to fructose. In order to produce fructose ata purity suitable for use as a food sweetener, it is necessary that theintermediate glucosone also be substantially pure.

Glucosone also has application in the production of mannitol andsorbitol. U.S. patent application Ser. Nos. 409,990, filed Aug. 20,1982, now abandoned, and 517,996 filed Aug. 1, 1983 entitled "Processfor the Production of Mannitol and Sorbitol", also assigned to theassignee of the present application and incorporated herein byreference, describe a method of producing a mixture of mannitol andsorbitol having an exceptionally high mannitol-to-sorbitol ratio. Themixture is prepared by reducing a solution of glucosone by catalytichydrogenation, using a nickel catalyst in the presence of hydrogen. Forproduction of a mannitol/sorbitol mixture suitable for use as a foodsweetener, substantially pure glucosone must be used.

Glucosone, being a relatively reactive compound, is also expected to beof use in other synthetic processes. For example, glucosone in aqueoussolution at pH between about 4.5 and about 8.5 (preferably between pH6.0 and 6.5) can be converted, by a novel pyranosone dehydratase enzymedescribed herein, to the antibiotic cortalcerone(2-hydroxy-6H-3-pyrone-2-carboxaldehyde hydrate, see Baute, et al.,Phytochemistry 15, 1753-1755 (1976); Baute, et al., Phytochemistry 16,1895-1897 (1977)).

This pyranosone dehydratase enzyme first catalyses the dehydration oftwo of the equilibrium forms of glucosone present in aqueous solution,alpha-D-arabino-hexosulopyranose and/or beta-D-arabino-hexosulopyranose,to 2,4-dihydroxy-6-hydroxymethyl-6H-3-pyrone (and tautomers thereof).The equilibrium forms of glucosone which are substrates for thepyranosone dehydratase both have a keto group at position 2 in thepyranose ring, an R configuration at carbon 4 in the pyranose ring (thesame stereochemical configuration as at carbon 4 in D-glucose), and anaxial hydrogen at carbon 3 (disposed with respect to the pyranose ringin the same way as the hydrogen at carbon 3 in D-glucose). Thisarrangement of keto oxygen at position 2, hydroxyl at position 4, andhydrogen at position 3 of a pyranose ring apparently provides thesubstrate specificity for catalytic dehydration (involving removal ofthe hydroxyl from position 4, and hydrogen from position 3) bypyranosone dehydratase. (A hydroxyl group at carbon 1 might also berequired for substrate specificity.)

For formal names of sugars and sugar derivatives used in the presentspecification, reference is made to R. Shallenberger, Advanced SugarChemistry: Principles of Sugar Stereochemistry, AVI Publishing Company,Inc., Westport, Conn., 1982, pp. 1-28. For determination ofconfiguratiion at chiral centers according to the R.S. system, referenceis made to J. Roberts and M. Casserio, Basic Principles of OrganicChemistry, W. A. Benjamin, Inc., New York, N.Y., 1964, pp. 592-593.

The intermediate, 2,4-dihydroxy-6-hydroxymethyl-6H-3-pyrone is named byanalogy with the formal name for cortalcerone, provided in Baute (1976),supra. The oxygen in the pyrone ring is at position 1, the keto-carbonat position 3 and the double bond between positions 4 and 5.

This intermediate has a highly strained structure. Its ring opensspontaneously in aqueous solution, forming4-deoxy-aldehydo-D-glycero-2,3-hexadiulose and tautomers thereof. Thecompound, and its tautomers, can recyclize between carbons 2 and 6 toform 3-deoxy-D-glycero-pentosulopyranose, 1-carboxaldehyde. Thiscompound, which has the required steric arrangement for catalytic actionby pyranosone dehydratase (an equitorial hydroxyl group on carbon 4, anaxial hydrogen on carbon 3, and a keto oxygen on carbon 2) can becatalytically dehydrated by the enzyme to remove the hydroxyl fromcarbon 4 and one of the hydrogens from carbon 3, to producecortalcerone, an antibiotic.

Another use of glucosone is described in U.S. Pat. No. 4,351,902,assigned to the assignee of the present application and incorporatedherein by reference. The patent describes the catalytic oxidation ofglucosone by glucose-1-oxidase to form 2-ketogluconic acid, which hasuses in food and other industries.

The other pyranosone which can be prepared as descfibed herein isxylosone. Xylosone can be reduced to xyulose by known methods,particularly by hydrogenation with molecular hydrogen in the presence ofa heavy metal catalyst (e.g. palladium or carbon), and by other methods,as described, for the reduction of glucosone by fructose, in U.S. Pat.No. 4,246,347 and Geigert, et al., Carbohyd. Res. 113, 159-162 (1983).Xyulose can be fermented to ethanol by common yeasts (e.g. Saccharomycescerevisiae), which are essentially incapable of fermenting xyulose, amajor by-product of biomass degradation. See Wang, et al., Biochem. andBiophys. Rsch. Commun. 94, 248-253 (1980); Chiang, t al., Appl. andEnviron. Microbiol. 42, 284-289 (1981). Thus, the methods and reagentsof the present invention for conversion of xylose to xylosone, withoutsubsequent significant enzymatic reaction of the xylosone, provide onestep in a process for utilizing xylose from biomass to make ethanol.

The novel pyranosone dehydratase enzyme, whose catalysis of thedehydration of glucosone to cortalcerone is described above and whoseproperties and isolation are further described below, also catalyzes thedehydration of xylosone in aqueous solution to compounds which would beexpected to have antibiotic properties similar to cortalcerone, andpotentially other properties which make them useful to the fungi whichproduce them, starting from xylose and passing through xylosone, underconditions of stress.

Xylosone exists in aqueous solution as an equilibrium mixture ofalpha,D-threo-pentosuloplyranose and beta,D-threo-pentosulopyranose.These compounds are the analogues of the alpha and beta anomers of theform of glucosone in solution on which the pyranosone dehydratase acts,as described above. The xylosones differ from the glucosones by theabsence of an hydroxymethyl group at carbon 5 in the pyranose ring. Thepyranosone dehydratase dehydrates both equilibrium forms of xylosone to2,4-dihydroxy-6H-3-pyrone (and tautomers thereof).

2,4-dihydroxy-6H-3-pyrone is, like its analog derived from glucosone, ahighly strained compound. It likely ring-opens to4-deoxy-aldehydo-2,3-pentadiulose and tautomers thereof. The compoundand any of the tautomers may be hydrated at the aldehyde group atposition-1.

Because 4-deoxy-aldehydo-2,3-pentadiulose lacks a sixth carbon atom, itcannot rearrange into a form with the steric requirement, describedabove, for the action of pyranosone dehydratase. Consequently, theenzyme dehydrates xylosone only once.

Both 2,4-dihydroxy-6H-3-pyrone, and its 6-hydroxymethyl analog haveabsorbance maxima in the UV at about 265 nm.4-deoxy-aldehydo-2,3-pentadiulose (or tautomers or aldehyde-grouphydrates of the compound) and 4-deoxy-aldehydo-D-glycero-2,3-hexadiulose(or tautomers or aldehyde-group hydrates of the compound) might absorbstrongly near 265 nm, but do not absorb significantly at any otherwavelength between about 200 nm and about 300 nm.3-deoxy-D-glycero-pentosulopyranose,1-carboxaldehyde (in anyconfiguration, whether hydrated or not) does not absorb in the UVbetween about 200 nm and about 300 nm. Cortalcerone, however, has anabsorption maximum only at about 230 nm, between 200 and 300 nm. Thus,adding pyranosone dehydratase enzyme to an aqueous solution of purexylosone results in increasing absorption at 265 nm and no absorbtion at230 nm as the xylosone is dehydrated to the 6H-3-pyrone. However, addingpyranosone dehydratase enzyme to an aqueous solution of pure glucosoneresults first in increasing absorption at 265 nm, as glucosone isdehydrated to the 6H-3-pyrone, and eventually in increasing absorptionat 230 nm and decreasing absorption at 265 nm as 6H-3-pyrone rearrangesto non-UV-absorbing 3-deoxy-D-glycero-pentosulopyranose,1carboxaldehyde, which in turn is dehydrated by the enzyme tocortalcerone.

The 5-ketofructose obtained in substantially pure form from sorbose,with the methods and reagents provided in the present specification, canbe converted to kojic acid, 3-oxykojic acid and 5-oxymaltol. See, e.g.Sato, et al., J. Agr. Biol. Chem. 31, 877-878 (1967) and 33, 1606-1611(1969). Kojic acid itself is an antibiotic and can be converted to theflavor-enhancing additives maltol and ethyl maltol. See Merck Index,10th Edition, p. 764 (1983).

The uses of mixtures of 2-ketogluconic and isoascorbic acids providedfrom delta-gluconolactone by the methods and reagents of the presentspecification are set forth in U.S. Pat. No. 4,351,902, supra.

The production of pyranosones, 5-ketofructose and mixtures of2-ketogluconic and isoascorbic acids from the corresponding pyranosesusing a pyranose-2-oxidase enzyme preparation from P. obtusus has beendescribed, e.g., Janssen and Ruelius, Biochim. et Biophys. Acta 167,501-510 (1968). Above-mentioned U.S. Pat. Nos. 4,246,347, 4,321,323,4,321,324 and 4,423,149 describe production of glucosone from glucoseusing glucose-2-oxidase enzyme preparations from various Basidiomycetes.

U.S. Pat. No. 4,423,149 is assigned to the assignee of the presentapplication and is incorporated herein by reference. Above-mentionedU.S. Pat. Nos. 4,351,902 and 4,423,149 describe production of mixturesof 2-ketogluconic and isoascorbic acids from deltagluconolacton usingglucose-2-oxidase enzyme preparations from Basidiomycetes. The prior artreferences in this area describe reactions using partially purifiedpyranose-2-oxidase (P20) derived from Polyporous obtusus, and carriedout at a preferred pH of about 7.0. For a variety of reasons which willbe explored below, these reaction methods inherently produce breakdownof the pyranosone products, reducing the yield and purity. The prior artmethods are also characterized generally by relatively short P2O halflives, due in part to a failure to control co-produced H₂ O₂ levelsadequately, as will be seen below.

It is therefore one general object of the present invention to provide amethod and reagent by which pyranoses can be enzymatically oxidized tothe corresponding pyranosones or related compounds with substantiallygreater yield and purity than is obtainable by prior art methods andreagents.

A particular object of the invention is to provide a method of producingsubstantially pure glucosone for use in making high-purity food additivesugars such as fructose, mannitol and sorbitol.

Yet another object of the invention is to provide a relativelyinexpensive and easily prepared P2O reagent which has enhanced stabilityunder the reaction conditions employed in the method of the invention.

Providing a reagent and method which are readily adaptable tocommercial-scale uses is still another object of the invention.

The method of the invention includes providing a P2O enzyme or enzymepreparation which is substantially free of measurablepyranosone-utilizing enzyme activity at a selected pH between about 4.4and 7.0 The P2O preparation is reacted with a pyranose substrate in thepresence of an H₂ O₂ -removing catalyst, such as catalase. A preferredH₂ O₂ -removing catalyst includes catalase which has been purified toremove substantially all glucose-1-oxidase activity. The reaction isperformed in the presence of oxygen at the selected pH between 4.4 and6.0.

One major pyranosone-utilizing enzyme which has been identified isreferred to herein as pyranosone dehydratase (PD), so called for itsability to dehydrate pyranosones such as glucosone and xylosone. Thisenzyme is present in partially purified P2O enzyme preparations obtainedfrom P. obtusus. The activity of the enzyme may be substantiallyeliminated, according to one aspect of the invention by (a) purifyingthe P2O preparation to remove PD; (b) heat-treating the P2O preparationto inactivate PD preferentially; and/or (c) carrying out the reaction atabout pH 4.4.

The invention also contemplates selecting, as a source of P2O, a fungalorganism which is capable of converting glucose to glucosone, evidencinga P2O enzyme, but which is incapable of converting glucose or glucosoneto cortalcerone, evidencing the absence of PD. Two preferred or9anismsinclude Coriolus versicolor and Lenzites betulinus.

In one embodiment of the invention, there is provided a reagent composedof a solid support and surface-attached P2O and catalase molecules, at aP20/catalase activity ratio of between about 10⁻⁵ to 10⁻³. The P2O isprepared to be free of substantially all pyranosone utilizing enzymecontaminants at a selected pH between about 4.4 and 6.0.

The invention also contemplates substantially pure P2O enzymes preparedfrom the above-mentioned sources, as well as cultures of fungi whichproduce enhanced amounts of P2O enzyme.

These and other objects and features of the invention will become morefully apparent from the following description of the invention, whenread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares the kinetics of heat inactivation of partiallyproteolyzed and intact forms of P2O and PD obtained from P. obtusus;

FIG. 2 shows the pH profile of P. obtusus P2O and PD;

FIG. 3A is an absorbance profile, at 192 nm, of HPLC-fractionatedreaction products obtained by reacting glucose, at pH 6.0, with apartially purified P. obtusus P2O enzyme preparation;

FIG. 3B is an absorbance profile, like that in FIG. 3A, of reactionproducts produced by reacting glucose at pH 6.0 with a P. obtusus P20enzyme which has been purified to remove glucosone-utilizing enzymecontaminants;

FIG. 3C is an absorbance profile, like that in FIG. 3A, of reactionproducts produced by reacting glucose at pH 6.0, with a P. obtusus P2Oenzyme preparation which has been heat-treated to inactivateglucosone-utilizing enzyme contaminants;

FIG. 3D is an absorbance profile, like that in FIG. 1A, of reactionproducts produced by reacting glucose, at pH 4.4, with the P2O enzymepreparation of FIG. A;

FIG. 3E is an absorbance profile, like that shown in FIG. 3A, where thereaction described in FIG. 3B was performed at pH 4.4;

FIG. 4 illustrates, in flow-diagram form, procedures used in providing aP2O enzyme in accordance with the invention; and

FIG. 5 shows an absorbance profile, at 192nm, of HPLC-fractionatedreaction products obtained by reacting glucose, at pH 5.0, with purifiedP. obtusus P2O in an unbuffered reaction medium.

DETAILED DESCRIPTION OF THE INVENTION

The invention includes providing a P2O enzyme, or enzyme preparationwhich is substantially free of pyranosone-utilizing enzyme contaminantswhich are measurably active at a selected pH between about 4.4 and 7.0.The P2O enzyme may be used in forming a reagent which also includes anH₂ O₂ -decomposing catalyst, such as catalase, at a P20/catalaseactivity ratio of less than about 10⁻³, i.e., an H₂ O₂ concentration ofless than about 10⁻³ M. The enzyme, or enzyme reagent, is reacted with apyranose substrate in the presence of oxygen, at the selected pH toproduce the corresponding, substantially pure product glucosone,xylosone, 5-ketofructose or mixture of 2-ketogluconic acid andisoascorbic acid. Preferred substrates are glucose and xylose; mostperferred is glucose. The reaction may be pH stable, even in anunbuffered reaction medium. Details of the invention will be describedbelow with particular reference to methods for providing a P2O enzyme,for producing a reagent containing the enzyme and an H₂ O₂ -decomposingcatalyst, and to reaction methods using the enzyme to producesubstantially pure pyranosones, particularly glucosone.

Providing a P20 Enzyme

As used herein, the terms "pyranose-2-oxidase" or "P20" designates anenzyme capable of catalyzing the conversion, in aqueous solution withoxygen as co-reactant and hydrogen peroxide as co-product, of thehydroxyl group at the second carbon position (in the cases of glucose,xylose and deltagluconolactone) or the fifth carbon position (in thecase of sorbose) of a pyranose to a keto group. The enzyme's preferredsubstrate is glucose, which is converted to glucosone; but the enzymealso is active in converting xylose to xylosone, sorbose to5-ketofructose and delta-gluconolactone to a mixture of 2-ketogluconicand isoascorbic acids.

Enzymes displaying such pyranose-2-oxidase activity may be obtained froma variety of microorganisms, molusca and red algae, as reported in theabove cited U.S. Pat. No. 4,246,347. Among the preferred sources of theenzyme are the white-rot fungi of the Basidiomycetes class, includingPolyporus obtusus, Coriolus versicolor, and Lenzites betulinusorganisms, expecially P. obtusus. These fungal organisms produce P2Oenzymes having a preferred substrate specificity for glucose and anadditional substrate specificity for sorbose, xylose anddelta-gluconolactone.

The amount of pyranose-2-oxidase enzyme produced by the selectedorganism may be increased by mutation and selection techniques.Typically, cells which have been prescreened for ability to producerelatively high P2O levels are exposed to a mutagenic agent, such asultraviolet light, at a preselected level. Following exposure to themutagen, the cells are individually selected for increased P2O activity.The mutation/selection procedure may be repeated to yield stillhigher-producing strains. Example I below details a mutation/selectionprotocol used to produce a P. obtusus strain which produces P2O at alevel which is about 5 times that of the unmutated P. obtusus cellsprior to the selection procedure.

A P2O enzyme preparation containing partially purified P2O is preparedby disrupting cells of the selected organism, and removing insolublecell material by centrifugation. Further enzyme purification is achievedby adding to the supernatant, an agent which produces precipitation ofselected supernatant proteins. Exemplary protein-precipitating agentsincluding salts, such as ammonium sulfate, solvents, such aspolyethylene glycol, and strong acids. The agent may be added at asingle concentration, or in a series of increasing concentrations. Theprecipitated proteins, which may include P2O, are removed bycentrifugation or filtration.

The P2O enzyme derived from P. obtusus and L. betulinus are preferablyfractionated by precipitating the enzymes by the addition ofpolyethylene glycol. Ammonium sulfate precipitation of the C. versicolorP2O enzyme is preferred. The enzyme precipitates may be resuspended in abuffer, incubated, and recentrifuged to remove insoluble proteinsremaining in the resuspended enzyme fraction.

Partially purified P2O enzyme preparations obtained from P. obtusus havebeen reported in the prior art. Janssen and Ruelius (1968) and U.S. Pat.Nos. 4,246,347; 4,351,902; and 4,423,149, supra. These patents disclosea partially purified pyranose-2-oxidase fraction obtained from P.obtusus, substantially according to the purification steps outlinedabove. The enzyme preparation is advantageous in that P. obtusus isknown to produce relatively high levels of P2O, and the enzyme can beobtained in large quantity from fermenting cells. The steps leading tothe partial enzyme purification can be carried out in commercial-scalequantities at relatively low cost, since only low-speed centrifugationsteps are involved.

However, a variety of studies in which the above partially purified P.obtusus P2O was used to convert glucose to glucosone have shown that theglucosone produced may be contaminated to a substantial degree withby-products. In one typical reaction study, reported in detail inExample II below, a partially purified P. obtusus P2O enzyme was reactedwith a solution of pure glucose in a 0.1M sodium phosphate buffer, pH6.0 in the presence of oxygen. H₂ O₂ generated in the reaction wasdecomposed by purified catalase also added to the reaction medium. Theglucose was completely used up after a 2 hour reaction time at 24° C.However, the reaction was carried out for 6 hours at room temperature,to accentuate glucosone conversion or degradation reactions whichcontribute to reaction impurities. The pH of the buffered reactionmedium remained constant over the reaction period.

The products were analyzed by high performance liquid chromatography(HPLC) as described in Example II. The eluate from the column wasmonitored at 192 nm, where both glucosone (which absorbs strongly below200 nm) and by-products (which absorb strongly about 230 nm), aredetectable. The absorbance profile of the reaction products, seen inFIG. 3A, shows a peak which co-elutes with authentic glucosone and amajor peak representing non-glucosone by-products. The relatively highratio of by-products to glucosone observed is somewhat exaggerated overwhat would be observed in an optimal-condition reaction, due to theextended time period of the reaction as noted above.

Heretofore, investigators in the field of the invention believed thatthe major cause of by-product formation observed in the enzymaticconversion of glucose to glucosone was due to chemical instability ofthe glucosone. Thus previous attempts to increase product yield andpurity of enzymatically produced glucosone centered on improvingreaction conditions to increase the chemical stability of the productformed, and to reduce the time during which product decomposition couldoccur.

An important feature of the present invention is the finding that amajor part of the non-glucosone by-products observed in an enzymaticglucosone-production reaction is due to the presence in the P2O enzymepreparation of one or more pyranosone-utilizing enzyme contaminants.Studies have led to the identification of one major, and perhaps sole,pyranosone-utilizing enzyme contaminant in P2O preparations from thefungal source, P. obtusus. The contaminating enzyme functions todehydrate pyranosones, and accordingly has been designated "pyranosonedehydratase." Of particular interest is the action of this enzyme onglucosone. Initial enzymatic dehydration of glucosone produces anunstable intermediate which has an absorption peak at about 265 nm. Theintermediate then rearranges to a stable pyranosone intermediate whichis further enzymatically dehydrated, by the same enzyme, to form theantibiotic cortalcerone, as discussed above. One of the functions of P2Oin some organisms, therefore, may be to produce glucosone for use in theenzymatic synthesis of cortalcerone by PD.

The PD enzyme acts also with xylosone to produce a product which alsoabsorbs with a peak near 265 nm. This product is the analog, (lacking ahydroxymethyl group at position 5 on the pyrone ring) of the firstintermediate formed from glucosone by the PD enzyme. Like the firstintermediate from glucosone, xylosone product intermediate may rearrangeby ring-opening. However, because it has six carbon atoms, thering-opened form of the intermediate from glucosone can undergo ringclosure to a form with the steric requirements for the PD enzyme act(i.e. dehydrate) again, resulting in cortalcerone. However, thestraight-chain form of the xylosone-derived intermediate, having 5carbons, cannot recyclize to a form that can be acted on further by thePD enzyme. The product of PD action on xylosone may also be anantibiotic. Thus, the role of P2O is some organisms might also be toprovide xylosone for subsequent dehydration with PD to provide anantibiotic for defensive purposes.

The method of the invention comprises, in part, providing a P2O enzymewhich is substantially free of pyranosone-utilizing enzyme contaminantswhich have measurable activity at a selected pH between about 4.4 and7.0. A first step in providing the enzyme involves selecting a suitableP2O source. Preferred P2O sources include microorganisms, andparticularly fungal organisms, which can be cultured in relatively largequantities in a relatively short time period. A microorganism, such as afungus, which produces P2O can be identified readily by the ability ofthe cells, when broken, to convert glucose to glucosone, as determined,for example, by the reaction of glucosone with triphenyltetrazoliumchloride (TTC) to give a strongly colored solution. By way ofillustration, a parental (unmutated) strain of P. obtusus was found toproduce between about 200 and 500 enzyme units per liter of fungalculture after a ten day culture period. Parental strains of C.versicolor and L. betulinus each produced between about 50 and 100 unitsper liter of fungal culture after a 7-day culture period.

Having identified a suitable microbial source of P2O, according to theabove screening procedure, the organism is then tested, in a broken-cellassay, for its ability to convert glucosone to cortalcerone, indicatingthe presence of PD. The conversion of glucosone to cortalcerone can bemonitored spectrophotometrically by following the reaction at thecortalcerone absorption peak at about 230nm, or at 265 nm, where anintermediate between glucosone and cortalcerone produced by PD fromglucosone absorbs. Alternatively, the disappearance of glucosone asmonitored, for example in the above TTC-colorometric assay, may be usedto screen for PD activity, and more generally, for glucosone-utilizingenzyme activity. Among the three Basidiomycetes listed above which werefound to produce P2O, only P. obtusus showed measurable PD activity. Theabove selection procedure is thus seen to identify two generalcategories of P2O producing organisms: one having the ability to convertglucose to glucosone and glucose or glucosone to cortalcerone, thelatter reaction evidencing the presence of PD, and a second groupcapable of converting glucose to glucosone, but incapable of furthermetabolizing glucosone to cortalcerone. These screening procedures couldalso be carried out using xylose and xylosone in place of glucose andglucosone, as xylosone can also be assayed using the TTC assay and isalso dehydrated by PD to a spectrophotometrically active product, whichabsorbs strongly at about 260 nm.

Where the organism selected includes both glucosone-producing andglucosone-utilizing enzymes, the invention further contemplatesproviding a P2O enzyme which is substantially free of all measurableglucosone-utilizing enzyme activity at a selected pH between about 4.4and 7.0. Initially a partially purified P2O preparation is obtained.Novel procedures for freeing the P2O enzyme preparation ofpyranosone-utilizing enzyme activity at such selected pH generallyinclude one of the following procedures:

(a) The P2O enzyme is purified to remove substantially allpyranosone-utilizing enzymes, and in particular PD, which are active ata pH of about 6.0 or below;

(b) The P2O enzyme is heat-treated to eliminate substantially allpyranosone-utilizing enzyme activity at a pH below about 6.0; and

(c) The P2O enzyme is utilized at a pH at or near about pH 4.4.

Although the three procedures discussed above will be described withparticular reference to a P2O enzyme fraction obtained from P. obtusus,it will be appreciated that the rationale underlying each of theprocedures will be applicable to P2O enzyme preparations obtained fromother sources.

Further purification of the enzyme preparation, in accordance withprocedure (a) above, is preferably carried out by ion-exchangechromatography. The chromatographic separation can be monitored readilyby assaying the eluted fractions for both P2O and PD activity. Theeluted samples are preferably assayed at a maximum pH of about 6.5. Theeluted samples showing highest P2O activity are combined, and wherenecessary, further separated from PD activity by molecular-sievechromatography. The reaction scheme used to purify P. obtusus P2Osubstantially to homogeneity is detailed in Example III below.

To test the ability of the purified P2O enzyme to produce substantiallypure pyranosone, a reaction study similar to the one described above,where the enzyme is used to convert glucose to glucosone in the presenceof purified catalase at pH 6.0, is performed. Example IV below givesdetails of experimental procedures used and results obtained, withreference to FIG. 3B. As can be appreciated from this figure, theglucosone produced, after a six hour incubation period at pH 6.0, issubstantially free of by-products, showing that most, if not all,glucosone breakdown at pH 6.0 is attributable to enzymatic breakdown.

Heat-treating the P2O enzyme preparation to inactivatepyranosone-utilizing enzyme contaminants, as in procedure (b), may bepreferred where the P2O is substantially more heat-stable thanpyranosone-utilizing enzyme contaminants. Heat inactivation can beperformed relatively simply and inexpensively by comparison withchromatographic purification techniques. In assessing the heat stabilityof the P2O enzyme, the effect of partial proteolysis and pH should beconsidered. For example, studies on the heat stability of P2O obtainedfrom P. obtusus show that the enzyme is readily proteolyzed followingcell homogenization, and that the partially proteolyzed enzyme is moreheat labile than the intact enzyme. Accordingly, it is advantageous toprepare the enzyme in the presence of proteolytic inhibitors such asphenylmethylsufonyl fluoride (PMSF) and/or ethylenediamine tetraaceticacid (EDTA).

The relative thermal stabilities of P. obtusus P2O isolated either inthe presence or absence of proteolytic inhibitors is shown in FIG. 1. Asseen here, a P2O preparation which is largely unproteolyzed loses onlyabout 20% of its total activity over a 90 minute incubation time at 65°C., at pH 5.0, while the partially proteolyzed enzyme loses more thanhalf its activity under similar conditions. Also as seen in this figure,purified P. obtusus PD lost substantially all of its activity after 15minutes of incubation at 65° C. Details of the heat-treatment studiesare found in Example V.

The effect of heat-treating a P2O enzyme preparation under selectedconditions can be determined by reacting the heat-treated enzyme withglucose under conditions substantially identical to those which havebeen described above with reference to FIGS. 3A and 3B. Example VIprovides details of a reaction employing a heat-treated P2O preparationobtained from P. obtusus in the absence of protease inhibitors. Thereaction products, fractionated by HPLC and monitored at 192 nm, gavethe absorption profile seen in FIG. 3C. The yield and purity of theglucosone produced using the heat-treated enzyme is substantiallyimproved over the results obtained using an untreated P2O preparation,and the improvement is expected to be still greater where proteaseinhibitors are added to the P2O preparation, and the length and/ortemperature of the heating step is increased to produce greaterinactivation of the pyranosone-utilizing enzyme(s).

The P2O obtained from P. obtusus is more resistant to heat inactivationthan the P2O obtained from either C. versicolor and L. betulinus. On theother hand the C. versicolor and L. betulinus P2O enzymes appear to bemore protease-resistant. It can be appreciated that purification toremove enzyme contaminants would be preferred for protease-resistant P2Oenzymes, whereas heat-treatment would be advantageous for moreheat-resistant P2O enzymes.

In procedure (c), pyranosone-utilizing enzyme activity in a P2O enzymepreparation is eliminated by utilizing of the enzyme at about pH 4.4.The method stems from the discovery that, for some P2O preparations,product-utilizing enzymes are largely inactive, but P2O has near maximalactivity, at this pH. The suitability of the method, when compared withP2O purification or heat treatment may vary with the P2O source. Toassess the value of this approach, pH activity profiles for P2O andglucosone-utilizing enzymes are determined. Example VII below detailsmethods used to determine the pH profiles of P. obtusus P2O and PD. Theresults, which are graphed in FIG. 2, show that P2O has a pH optimumbetween about 4.5 and 5.5, that the pH optimum of PD is about 7.5, andthe PD retains less than about 5% of its optimal activity at pH 4.4.

The ability of a P2O preparation to produce substantially pure productat pH 4.4 can be tested in the already-described reaction systeminvolving the enzymatic conversion of glucose to glucosone. Details of atypical experiment to determine glucosone purity and yield at pH 4.4,using the above P. obtusus P2O preparation, are given in Example VIII.HPLC analysis of the reaction products monitored at 192 nm is shown isFIG. 3D. Here it is seen that performing the reaction at a pH at whichat least one major glucosone-utilizing enzyme (PD) is substantiallyinactive leads to the production of a substantially pure glucosone. Theresults indicate that glucosone-utilizing enzyme contaminants, if any,other than PD are also substantially inactive at pH 4.4.

It can be appreciated that the above methods used to eliminateproduct-utilizing enzyme contaminants in the pyranose-2-oxidase enzymepreparation can be combined, where desired, to increase product purityand yield. For example, the P2O preparation may be fractionated toremove product-utilizing enzymes and the purified P2O then used in areaction which is carried out at pH 4.4. Example IX below describes sucha reaction for producing glucosone from glucose. The HPLC analysis ofthe reaction products is shown in FIG. 3E. The glucosone produced in thereaction is greater than 99% pure, as determined by relative peak areasobtained following HPLC, as measured by both refractive index andultraviolet absorption at 192 nm.

According to another embodiment of the invention, a P2O enzyme which issubstantially free of pyranosone-utilizing enzyme contaminants may beprovided by selecting, as a source of the P2O enzyme, a microorganismwhich is capable of converting glucose to glucosone, but is unable toconvert glucose or glucosone to cortalcerone. This selection procedureidentifies microorganisms which synthesize P2O but little or no PD. Theorganism is screened for the ability to convert glucose to glucosone andglucosone to cortalcerone by conventional assay methods, such as thosereferred to above. Example X describes specific procedures used inselecting two fungal organisms, C. versicolor and L. betulinus, whichproduce moderate levels of P2O, but which lack the ability to convertglucosone to cortalcerone.

A purified or partially purified P2O enzyme is obtained from theselected source lacking measurable PD activity. Generally, the P2Opurification procedure comprises (1) obtaining a cell supernatantfraction containing the P2O enzyme, and (2) selectively precipitatingthe P2O with a protein-precipitating agent such as ammonium sulfate orpolyethylene glycol. Where necessary, the P2O enzyme may be furtherpurified by chromatographic techniques, such as ion-exchange and/ormolecular sieve chromatography.

Examples XI and XII below outline purification procedures used to obtainsubstantially pure P2O from C. versicolor and L. betulinus,respectively. P2O obtained from L. betulinus was assayed for its abilityto convert glucose to glucosone in a reaction containing 20 mM glucose,catalase, and oxygen, in a suitable buffer. After a two-hour reactionperiod, the products were fractionated by HPLC as monitored at 192 nm.Products analysis showed that the glucosone produced was substantiallypure.

FIG. 4 presents, in flow-chart format, a general procedure for providinga P2O in accordance with the invention, based on the findings presentedabove. The first step in the procedure is to identify microbial P2Oproducers, such as the three fungal sources of P2O discussed herein.

Considering the scheme shown in FIG. 4, the organism may be carriedthrough one or more mutation and selection steps, such as described inExample I, to produce strains which produce elevated levels of P2Oand/or enhanced P20/PD ratios. An especially advantageous strainobtained by mutagenesis and selection is P. obtusus strain AU124,cultures of which have been deposited at NRRL under No. 15595.

A partially purified P2O enzyme is obtained by batch purificationtechniques which preferably involve selective protein precipitation andcentrifugation steps only. The partially purified P2O is then assayedfor pyranosone-utilizing activity, e.g., by the ability of the enzymepreparation to convert glucose to substantially pure glucosone at about6.0. If no pyranosone-utilizing activity is observed in this indirectassay, or in a direct PD activity assay monitored at either 230 or 265nm, the preparation may be suitable for use as a "final" P2O enzyme inaccordance with the invention. Such might be the case, for example,where the P2O preparation is obtained from a selected source such as C.veriscolor or L. betulinus which does not show measurable PD activity oninitial screening.

Where, as with the P2O enzyme preparation obtained from P. obtusus,relatively high levels of product impurities are observed, e.g., by HPLCanalysis, evidencing high PD activity at pH 6.0, the contaminatingenzyme activity is substantially eliminated by one or more of the threeprocedures described. In one procedure, the enzyme preparation isutilized at a pH at which P2O activity is optimal or near-optimal, butat which pyranosone-utilizing enzyme(s) are largely inactive. In asecond procedure, the P2O preparation is heat treated to inactivatepyranosone-utilizing enzymes preferentially. Both methods are suited tolarge-scale processes and are rapid and inexpensive, making themattractive for commercial-scale preparation of a P2O enzyme.

Where necessary, the P2O preparation can be further purified to removeproduct-utilizing enzyme contaminants. The findings presented abovesuggest that PD is the major, if not the only, pyranosone-utilizingenzyme in the P. obtusus P2O enzyme preparation. Therefore, thepurification method can focus on removal of PD only, simplifying theprocedure.

Preparing Immobilized P20 Reagent and Controlling Peroxide

The conversion of a pyranose to the corresponding pyranosone by P2O inthe presence of oxygen is accompanied by the stoichiometric productionof H₂ O₂. The H₂ O₂ produced in the reaction may promote oxidativedegradation of the product. Studies performed in support of the presentapplication indicate that nonenzymatic breakdown of glucosone to acidicproducts may be accelerated at H₂ O₂ concentrations above about 0.2 mM.More importantly, elevated H₂ O₂ concentrations produce relatively rapidinactivation of the P2O enzyme. The susceptibility of P2O toinactivation by elevated concentrations of H₂ O₂ in a continuous-flowreactor is described below in Example XIII. Briefly, fourcontinuous-flow reactors containing different P2O/catalase activityratios were run in the presence of pure glucose. The half-life of theP2O enzyme in each reactor was determined from the observed change inthe amount of glucosone eluting from the reactor over a several-dayreaction period. The measured half-lives of P2O expressed in arbitraryhalf-life units, are given in Example XIII, Table 1. As will be seenfrom the table, the presence of catalase, at a P2O/catalase activityratio of less than about 10⁻³ and preferably less than about 2×10⁻⁴(i.e., at an H₂ O₂ concentration of less than about 0.2mM) extends themeasured P2O half-life 30-60 fold with respect to that of P2O reactedwith glucose in the absence of catalase.

The invention contemplates the use of a variety of peroxide-destroyingcatalysts to maintain the H₂ O₂ concentration of the reaction medium ator below a desired level. These reagents include enzymes, such ascatalase, or metal catalysts, such as platinum. A preferred catalystincludes catalase which has been freed of glucose-utilizing enzymes,such as glucose-1-oxidase, which are active in the pH 4.4 to 6.5 range.The catalase may be obtained from conventional sources, such asAspergillus niger.

Since P2O is both the site of H₂ O₂ production, and a target of H₂ O₂inactivation, it is important to prevent locally high H₂ O₂ transients,in the vicinity of the P2O, as well as the buildup of H₂ O₂ in the bulkphase. To this end, the catalyst is preferably disposed in closephysical association with the P2O, preferably by coupling the P2O to asolid-support substrate having a surface-associated H₂ O₂ -decomposingcatalyst. Intimate association between the oxidase enzyme and thecatalyst on such a solid support is important particularly in a reactorsystem in which the medium is relatively unagitated, as in a packedreactor column.

A reagent constructed according to the invention includes a solidsupport and a surface array of molecules of P2O and an H₂ O₂-decomposing catalyst. The support may include a broad range ofsurface-treated or untreated organic and inorganic supports. Includedamong these are polyacrylamide, ethylene maleic acid copolymers,methacrylic-based polymers, polypeptides, styrene-based polymers,agarose, cellulose, dextran, silica, porous glass beads, charcoal,hydroxyapatite and aluminum or titanium hydroxide.

Methods for attaching enzymes and non-protein catalysts to solidsupports are well known and include adsorbing the enzyme to the support,or coupling by covalent attachment. A reagent containing both P2O andcatalase is preferably formed by sequentially coupling catalase, thenP2O to the support surface. Where the H₂ O₂ decomposing catalyst is aninorganic catalyst, such as platinum, the reagent preferably is formedas a catalyst-coated solid support to which the P2O is attached.

In a commercial-scale reaction, the activity ratio of P2O to catalyst inthe reagent is selected to make the operation most cost effective. Amongthe factors to be considered are the relative costs of the P2O andcatalyst, the efficiency of the reaction at different P2O/catalystactivity ratios, the effect of H₂ O₂ inactivation of the P2O and/orcatalyst on the purity of the reaction product, and the purity andstability the product is required to have for the application(s) inwhich it is to be employed. As noted above, a P2O/catalase activityratio of about 10⁻³ or less produces a relatively long P2O half-life ina continuous-reaction system.

Control Of Reaction pH

This section examines pH control in the method of the invention. Asindicated above, the reaction is carried out at a selected pH between4.4 and 7.0. The upper pH limit of 7.0 is based on two independentconsiderations: First, the P2O enzymes whose characteristics arereported herein, including the P2O obtained from P. obtusus, L.betulinus and C. versicolor, show pH optima generally in the pH 4.0 to6.0 range, whereas the pyranosone-utilizing enzyme (PD) from P. obtususappears to be most active near pH 7.5.

Secondly, studies indicate that pure glucosone undergoes relativelyrapid, nonenzymatic breakdown above a pH of about 7.0. In oneexperiment, a 0.5% solution of highly purified glucosone (prepared byenzymatic conversion of glucose by a purified P. obtusus P2O enzyme atpH 4.4) was brought to pH 7.0 in a buffered solution. Immediatelythereafter, the solution was scanned spectrophotometrically in the UVrange between 200 and 350 nm, at one minute intervals. A slight peakobserved initially at 310 nm continued to increase appreciably at eachone minute interval, ultimately increasing several-fold over a fiveminute period, indicating rapid, non-enzymatic glucosone breakdown to acompound absorbing at 310 nm. In a similar experiment involving a 0.5%solution of glucosone at pH 6.0, no initial peak at 310 nm was observednor did one form during the several-minute period during whichone-minute interval scans were performed. Thus, non-enzymatic breakdownof glucose can be significantly reduced by maintaining a reaction pH ofabout 7.0 or below.

At pH's less than about 4.4, P2O and catalase activities decreaseappreciably. It is seen in FIG. 2, for example, that P. obtusus P2Oshows a sharp dropoff in activity between about pH 4.4 and pH 3.0.Similarly, catalase obtained from A. niger shows a sharp decline inactivity below about pH 4.4.

Heretofore, enzymatically catalyzed glucose-to-glucosone reactions havebeen characterized by a gradual drop in pH during the course of thereaction. Previous studies on the chemical stability of glucosone byEricsson, et al., Cellulose Chem. Technol., 7, 581 (1973), and byLindberg, et al., Acta Chem. Scand., 6, 1782 (1968), indicate that theobserved pH drop may be due to nonenzymatic glucosone breakdown to sugaracids.

The pH drop observed in glucose-to-glucosone reactions carried out byprior art methods necessitates carrying out the reaction in a strongbuffer and/or monitoring the reaction pH and adding a base periodicallyto offset acid formation. The reactions described above with referenceto FIGS. 3A-3E employ 0.1 M phosphate (pH 6.0) or citrate (pH 4.4)buffers, which were effective in maintaining the reaction pH constantover the 6-hour reaction periods used. However, in these reactionsrelatively low concentrations of substrates were used. Where higherconcentrations of products are being generated in the reaction, strongerbuffers would be required. However, a problem exists with the use ofstronger buffers in that they may be incompatible with subsequentreactions used to convert the pyranosone product, such as glucosone, tothe desired end product, such as fructose, mannitol or sorbitol. Inaddition, high salt concentrations are undesirable in food-gradeproducts such as those. Finally, experiments conducted by the inventorshave also shown that high-strength buffers may inhibit P2O activity.

Attempts to control reaction pH by the periodic addition of base havenot been entirely successful. The above-referenced studies by Ericsson,et al. and Lindberg, et al., show that glucosone breaks down underalkaline conditions. Therefore, in order to reduce glucosone breakdown,the base must be added in a manner which minimizes local regions of highpH in the solution. The present inventors have found that controllingthe reaction pH by adding NaOH periodically to maintain reaction pHleads to more glucosone breakdown than where a buffer such as lM sodiumbicarbonate or lM TRIS-HCl is used. Introduction of the buffer withrapid mixing further reduces glucosone breakdown, presumably by reducinglocally-elevated pH levels which occur when the buffer is added. It wasnoted above that even pH's in the range of 7.0 can lead to relativelyrapid nonenzymatic glucosone breakdown. Ideally therefore, where highpurity and yield are desired, one would like to avoid adding base to thereaction to offset the buildup of acidic by-products.

Heretofore, the enzymatic breakdown of enzymatically produced pyranosoneto acidic by-products has not been considered as a primary mechanism ofacid generation in reactions of the type considered herein. It is animportant finding of the present invention, therefore, that reactionsconducted in accordance with the method of the invention, in whichsubstantially all pyranosone-utilizing enzyme activity is eliminated, donot show the characteristic drop in pH seen in glucosone-producingreactions known in the prior art. Accordingly, a reaction carried outusing the method of the invention may be performed in an unbuffered orweakly buffered solution, without periodic addition of base, overextended reaction periods.

A reaction in which glucose is converted to glucosone by a substantiallypure P2O, in the presence of purified catalase, without pH control, isdescribed in Example XIV below. The reaction, involving the conversionof a 6% solution of glucose to glucosone, is carried out for 3 hours atroom temperature in an unbuffered aqueous medium at pH 5.0. The pHremained constant during the reaction. HPLC analysis of the reactionproducts indicate that the glucosone produced was about 99% pure.

From the foregoing, several important advantages of the invention can beappreciated. The method of the invention provides a significantimprovement in purity and yield over prior art methods for converting apyranose such as glucose to a pyranosone, such as glucosone. The purityand yield improvement is especially important to the success ofcommercial reactions in which a pyranosone such as glucosone is used asan intermediate in the production of a food product.

Another important advantage in commercial production is that a suitableP2O preparation can be provided without involved enzyme purificationprocedures. In two P2O preparation methods described herein, nochromatographic purification techniques are used. In another,purification may involve selective removal of a pyranosone dehydrataseenzyme only. In yet another, the source of P2O selected is one whichlacks the major, and perhaps only, pyranosone-utilizing enzyme, PD.

Under the reaction conditions employed, generation of acidic by-productsis substantially eliminated. The reaction can therefore be carried outover a several-hour period in an unbuffered reaction medium withoutaddition of base.

A reagent constructed according to the invention includes a solidsupport having arrayed thereon molecules of P2O and H₂ O₂ -removingcatalyst, at an activity ratio between about 10⁻⁵ and 10⁻³. This reagenthas a half life which may be 30 to 60 times that of the P2O enzyme in areaction where H₂ O₂ levels are not controlled.

The following examples describe particular embodiments of making andusing the invention, but are not intended to limit the scope of theinvention.

EXAMPLE I Selection of a P. obtusus strain which produces high levels ofpyranose-2-oxidase

This example describes the method used to produce a strain of P. obtususwhich produces a level of P2O several times higher than that of the P.obtusus culture (ATCC 26733; NRRL 15592) from which the strain wasderived. Mycelial pods of P. obtusus, ATCC No. 26733 also deposited byus at NRRL under No. 15592, and at CMCC under No. 0181, were grown onyeast/malt extract agar slants at pH 6.4, using standard yeast maltextract available from Diffco Laboratoraies (Detroit, Mich.) or agarprepared by mixing 3 g yeast extract, 3 g malt extract, 5 g peptone, 10g glucose and 20 g agar in 1 1 of distilled water and autoclaving understandard conditions. The slant-grown organism was used to inoculate ayeast/malt extract medium and was grown for 7 days on a rotary shaker at200 rpm, at room temperature. An inoculum from the liquid culture wasregrown in yeast/malt extract medium under similar conditions.

The culture material was disrupted by blending at high speed in ablender at 4° C. to break up mycelial filaments into small fragments,and these were plated out on yeast-malt extract agar plates. The platedcolonies were transferred to liquid-medium growth flasks, and grown upunder standard conditions. The cells were disrupted, and the contents ofthe cells assayed for the ability to convert glucose to glucosone.Glucosone production was determined using the triphenyltetrazoliumchloride assay, which gives a strong colorimetric reaction in thepresence of glucosone (Mattson, et al., Anal. Chem. 22:182 (1950).

Those cells which were the highest producers of glucosone were thenrecultured according to the method used initially. The resulting P.obtusus culture was composed of a substantially uniform population ofrelatively high P2O producers, denoted herein as the T strain. P.obtusus T strain cultures have been deposited by us at NRRL under No.15594. They are also deposited at CMCC under No. 1421.

The cultured T strain was washed in buffer and disrupted by blending, asabove. The disrupted culture material was added to a petri dish, in anamount just sufficient to cover the bottom of the dish, and irradiatedwith a General Electric 15W germicidal ultraviolet lamp for about 1-2minute, while stirring the material rapidly with a magnetic stirrer toproduce even irradiation of the cells. The irradiation time and fluxdensity were varied, the flux density by varying the distance betweenlamp and dish, to produce a survival rate of about 1-5%, based on numberof colonies observed by agar plating before and after irradiation.Following mutagenesis with UV light, the cells in the flask were platedout on yeast/malt extract agar plates, and the colonies formed on theplates were grown up in liquid medium and assayed, as above describedfor the T strain, for their ability to convert glucose to glucosoneenzymatically in a broken-cell assay.

A culture of one mutagenized, high-producer strain selected from amongthe mutagenized cell material produced about five times as much P2O,based on enzyme units per liter of culture material after six days, asthat produced by the parental ATCC #26733 strain. Cultures of thehigh-producer strain, designated AU124, have been deposited at the NRRL,under No. 15595. Cultures of the strain are also deposited at CMCC underNo. 1500.

EXAMPLE II P2O enzyme preparation from P. obtusus

P. obtusus, strain AU124 was grown on yeast/malt extract agar slants atpH 6.4. The slant-grown culture was used to inoculate a yeast/maltextract medium and was grown for 8 days on a rotary shaker at 200 rpm atroom temperature. The culture was vacuum filtered through number 541Whatman Paper.

The mycelia obtained from 400 ml of culture were washed twice with 0.05M sodium phosphate buffer, pH 7.5, containing 10 mM EDTA and 50 ug/mlfresh phenylmethysulfonyl fluoride (PMSF). The mycelia were homogenizedfor 2 minutes in a Waring blender with 70 ml of 0.05 M sodium phosphatebuffer pH 7.5, containing the above concentrations of proteaseinhibitors. The homogenate was centrifuged at 6,000 rpm for 20 minutesand the supernatant, containing the P2O activity, was decanted andplaced in a 500 Erlenmeyer flask. Polyethylene glycol was added to afinal concentration of 19 grams per 100 ml. The solution was stirred andthen allowed to stand at 40 minutes at 4° C. The precipitate wasrecovered by centrifugation at 10,000 rpm for 20 minutes.

The supernatant from the centrifugation step was decanted and discarded,and the pellet, containing the P2O enzyme activity, was resuspended byvortexing in 30 ml of 0.025 M sodium phosphate buffer, pH 7.5,containing 0.1 M sodium chloride and protease inhibitor(s) at theabove-mentioned concentrations. The purification was carried out asrapidly as possible at 4° C. to minimize proteolysis of the P2O enzyme.

The suspension was allowed to stand for 30 minutes, during which time aprecipitate formed, then centrifuged at 10,000 rpm for 20 minutes. Theresulting clear, yellow supernatant had a specific activity of about 4units per milligram of protein, as compared with about 0.5 units permilligram of protein in the supernatant fluid of the crude, clearedlysate. The enzyme so obtained is also referred to herein as apartially-purified P2O enzyme preparation.

The P2O enzyme preparation was tested for its ability to convertsubstantially pure glucose to glucosone at pH 6.0 in the presence ofoxygen and catalase. The catalase used was a highly purified A. nigercatalase obtained from Fermco, Inc. (Elk Grove, Illinois) (Lot No. 4927,154U/mg). Glucose-1-oxidase contaminants in the catalase were removed bypurification using DEAE chromatography. One gram of catalase waspurified by elution from a 5×12 cm column using a 0-150 mM NaCl gradientin 20 mM sodium acetate (pH 4.7). The bulk of the catalase activityeluted in a peak which was well separated from glucose-1-oxidaseactivity. Catalase was monitored by absorption of its heme group at 404mM.

Conversion of a 2% glucose solution in 0.lM sodium phosphate, pH 6.0, toproducts was carried out in a total volume of 500 microliters in a13×100 mm glass test tube. The reaction solution contained 0.46 units ofthe partially purified P2O enzyme preparation, and purified catalase.The tubes were shaken at 250 rpm at 25° C., 90% relative humidity, for 6hours and the reaction terminated by freezing.

The samples, after thawing, were analyzed by HPLC, using a Watersmicrobondapack carbohydrate column (Waters Corp.; Milford, MA),monitored by refractive index and ultraviolet absorption at 192 mM. Theresults are shown in FIG. 3A. Glucosone was identified by its elutionposition compared with a known glucosone standard. As seen in FIG. 3A,the partially purified P2O preparation produces substantial quantitiesof one or more non-glucosone by-products.

EXAMPLE III Purification and characterization of P. obtusus P2O

The P2O enzyme preparation from Example II was purified further usingDEAE chromatography in 30 mM TRIS-HCl, pH 8.5, containing 2 mM EDTA and10 ug/ml PMSF. The sample was eluted from a 5×16 cm column using agradient of 0-300 mM NaCl. P2O activity was monitored using theorthodianisidine colorometric assay for the peroxide co-product,essentially as described by Ruelius et al., Biochim et Biophys. Acta167, 493-500 (1968). P2O activity eluted at about 50 mM NaCl. Fractionscontaining the peak activity were pooled and concentrated to 0.5 ml byultrafiltration using Amicon filters obtained from Amicon Corp.(Danvers, MA). Biogel P300 was obtained from Biorad Laboratories(Richmond, CA). The sample was further purified by molecular sievechromatography using a 1.5×95 cm Biogel P300 column in 100 mM sodiumphosphate, pH 7 buffer. The P2O activity eluted at a sharp peakcorresponding to an apparent native molecular weight of about 290,000daltons.

The purity of the P2O fraction which eluted from the P300 column wasexamined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), according to a standard procedure. The P2O sample showed asingle band having a migration distance corresponding to a molecularweight of about 68,000 daltons. The enzyme was judged to be about 98%pure, based on the relative intensity of the faint contaminant proteinbands with respect to the major band corresponding to P2O subunit.

The purified P2O enzyme had a specific activity of about 8.6 units permilligram, representing an approximately 22-fold purification over theactivity of P2O seen in cleared lysates. The amount of enzyme recoveredwas about 40% of the total starting material.

The approximate 290,000 dalton molecular weight of the native P2O enzymeand its approximate 68,000 subunit molecular weight suggest that theprotein is a homotetramer. The enzyme appears to contain one covalentlybound flavin molecule per subunit (detectable as UV-fluorescence in the68,000 dalton SDS-PAGE band), and has absorption maxima at 276, 357 and456 nm. The enzyme has a sedimentation coefficient of about 11.4, asdetermined by sucrose gradient ultracentrifugation. The relativeactivities of the enzyme, on D-glucose, L-sorbose, D-xylose, andD-gluconolactone are 100: 80: 48: 12, respectively, at about 1% (w/v)substrate concentration. As noted above with respect to FIG. 2, theenzyme has a broad pH optimum of between about 4.5 and 5.5.

EXAMPLE IV Glucosone production with purified P2O

The purified P2O obtained in accordance with Example III was used toconvert glucose to glucosone in a reaction substantially identical tothat described in Example II. Briefly, 0.46 International Units of theenzyme were mixed in a 0.5 ml reaction medium containing 2% glucose in0.1 M sodium phosphate, pH 6.0, and purified catalase. The reaction wasperformed at 25° C. in the presence of air, at 90% relative humidity,for 6 hours. The reaction products were analyzed by HPLC, with thematerial eluted being monitored by ultraviolet light absorption at 192mm. The absorbance profile of the material produced in the reaction isshown in FIG. 3B. Here it is seen that substantially all of the materialproduced is in the prominent peak identified as glucosone, with verylittle by-product material being formed. A comparison of FIGS. 3B and 3Ashows the importance of removing glucosone-utilizing enzymecontaminants, including primarily, if not solely, PD, in improving boththe yield and purity of the glucosone produced by P2O.

EXAMPLE V Heat-treating the P 2O enzyme preparation

A P2O enzyme preparation was prepared from P. obtusus AU124substantially in accordance with the method described in Example II. TheP2O enzyme in the preparation was substantially unproteolyzed by virtueof the protease inhibitors used during the preparation steps. A secondP2O enzyme preparation studied was prepared in substantially the sameway, except that no protease inhibitor was used in any of thepreparation steps. When the P2O of this preparation was purifiedsubstantially to homogeneity, it was found to have variable subunitmolecular weights, principally molecular weights of about 65,000daltons, 34,000 daltons, 30,000 daltons, and approximately 4,000daltons, indicating partial proteolysis of the enzyme.

Heat-treatment of each enzyme preparation was carried out at pH 5.0 in50 mM sodium citrate, 1 mM EDTA, at 65° C. The P2O activity in eachpreparation was measured at the beginning of the heat-treatment andafter 15, 30, 60 and 90 minutes of heating at 65° C. P2O activity wasdetermined at 25° C. by oxygen consumption in the presence of glucose,using an oxygen electrode according to known methods. In FIG. 1, it isseen that P2O, which is largely unproteolyzed ,loses less than about 10%of its activity after incubation at 65° C. for 15 minutes, and about 20%of its original activity after 90 minutes of heat-treatment. Bycontrast, the partially proteolyzed P2O loses about 30% of its activityin the first 15 minutes and more than half of its activity after 90minutes of heating.

PD was removed from the nonproteolyzed P2O enzyme fraction and purifiedsubstantially to homogeneity. Briefly, the PEG-precipitated P. obtususP2O enzyme preparation was fractionated using DEAE chromatography, pH8.5. The PD enzyme was identified by its ability to convert glucosone toproducts having optical absorption at 230 and 265 nm. To remove minorcontaminants, the column fractions containing maximal PD activity wererechromatographed on a smaller DEAE column, using the same conditions.Peak enzyme fractions from the second DEAE column were further purifiedon a carboxymethyl sepharose column (Pharmacia CL-6B) and elutedtherefrom, at pH 5.5, by a 0-200 mM NaCl gradient in 50 mM sodiumacetate, pH 5.5. The resulting partially purified PD was diluted into 50mM sodium acetate, pH 5.0, containing 1 mM EDTA and a small amount ofpurified P. obtusus P2O and heat-treated at 65° C. for time intervalsranging from 5 minutes to 30 minutes. When the enzyme solution wasreadjusted to pH 6.0 and reassayed at 22° C., the PD was found to havelost substantially all of its activity after 15 minutes of heating at65° C. The P2O in the same solution retained substantially all of itsactivity after the same 15 minute treatment, similar to what is shown inFIG. 1.

EXAMPLE VI Glucosone production using heat-treated P2O enzymepreparation

The partially proteolyzed P2O enzyme preparation from Example V washeat-treated, at pH 5.0, for 15 minutes at 65° C. The heat-treatedfraction was reacted with 2% glucose under conditions identical to thosedescribed in Example II. The reaction products were separated by HPLCand monitored at 192 nm, with the results seen in FIG. 3C. It can beseen from a comparison of FIGS. 3C and 3A that heat-treating the P2Oenzyme preparation produced a substantial increase in the yield ofglucosone, and a corresponding decrease in by-products.

EXAMPLE VII P. obtusus P2O and PD pH Profiles; Molecular Weight of PD

P2O and PD from P. obtusus, strain AU124, were isolated in accordancewith the methods described in Examples III and V, respectively. Theactivity of the P2O enzyme, as determined by the rate of O₂ consumptionin the presence of glucose, was measured over a pH range from about 3.0to 7.0. The pH profile of P2O, indicated by the dashed line in FIG. 2,shows that the enzyme pH optimum is between 4.5 and 5.5. Similarly, thepH profile of the purified PD, as determined by its ability to convertglucosone to the product which absorbs at 265 nm, was measured over a pHrange from about 4.0 to 10.0. The PD pH profile, indicated in solidlines in FIG. 2, shows that the enzyme has a pH optimum of about 7.5,and displays less than about 5% of its optimal activity when assayed atpH 4.4.

PD-containing fractions with peak activity eluted from the carboxymethylsepharose column as described in Example V, were pooled and concentratedto 0.5 ml, using Amicon PM30 ultra-filtration, and then sized accordingto native molecular weight by molecular sieve chromatography using a1.5×95 cm Biogel P300 column, with 100 mM sodium phosphate buffer, pH7.0. PD activity eluted as a sharp peak at an apparent native molecularweight of about 200,000 daltons. Using peak and side fractions from theBiogen P300 sizing column, PD was further analyzed by SDS-PAGE accordingto a standard procedure. The sample showed a single band with molecularweight of about 98,000 daltons. PD from P. obtusus is thus apparentlycomposed of two identical subunits.

EXAMPLE VIII Glucosone production with the P2O enzyme preparation at pH4.4

The enzymatic conversion of glucose to glucosone by the partiallypurified P2O enzyme preparation from Example II at pH 4.4 was studied.The reaction conditions were substantially identical to those describedin Example II, except that the reaction was carried out at pH 4.4, usinga 0.1 M sodium citrate buffer rather than the 0.1 M sodium phosphatebuffer used for the pH 6.0 reactions described in Examples II, IV andVI. The reaction products were analyzed by HPLC, and monitored at 192nm, with the results shown in FIG. 3D. The pH remained substantiallyunchanged over the 6 hour reaction period. As seen from the absorbanceprofile in FIG. 3D, performing the reaction at pH 4.4 leads to a veryhigh yield and purity of glucosone, and almost complete suppression ofby-products formed in the reaction.

It was seen in Example VII above that the PD enzyme contained in the P.obtusus P2O enzyme preparation is largely inactive at pH 4.4. The factthat by-product formation is largely suppressed at pH 4.4 indicates that(1) PD is the major, if not sole, glucosone-utilizing enzyme contaminantin the P2O enzyme preparation and/or (2) the PD pH profile, andparticularly its low activity at pH below about 4.4, is typical of otherglucosone-utilizing enzyme contaminants in the P2O preparation.

EXAMPLE IX Glucosone production with purified P2O at pH 4.4

The present example demonstrates the high product yield and purityachievable in a glucose-to-glucosone reaction using a substantiallypurified P2O enzyme and carrying out the reaction at pH 4.4. Thereaction conditions used were substantially those used in Example VIII,except that P2O enzyme derived from P. obtusus AU124 and purified inaccordance with Example III was used instead of the partially purifiedP2O enzyme preparation from Example II. The products were analyzed byHPLC, as monitored at 192 nm, with the results shown in FIG. 3E. Theresults indicate that substantially no by-products were formed duringthe 6 hour reaction period. The purity of glucosone produced in thereaction was estimated to be greater than 99%, based on the relativepeak areas of glucosone and by-products.

EXAMPLE X Selection of P2O-producing Microorganism Lacking PD EnzymeActivity

This example illustrates another embodiment of the method of theinvention for providing a P2O enzyme which is substantially free ofpyranosone-utilizing enzyme contaminants at a selected pH between about4.4 and 6.5.

A number of fungal species have been found to be capable of convertingglucose to glucosone. These include Aspergillus flavus (C. Berkeley,Biochem. J. 27, 1357 (1933); Aspergillus parasiticus (C. Berkeley,Biochem. J. 31, 1033 (1937); Polyporus obtusus (Janssen and Ruelius(1968), supra); Corticium caeruleum (Baute, et al. (1977), supra); andOudemansiella mucida (Volc, et al., Coll Czech. Chem. Commun. 45, 950(1980). The ability of a microorganism to convert glucose to glucosonepresumably requires that the microorganism possess an enzyme or enzymesystem with glucose-2-oxidase activity. The P. obtusus and O. mucidaspecies have been shown to have an enzyme or enzyme system withpyranose-2-oxidase activity. Janssen and Ruelius (1968), supra; Volc, etal. (1980), supra. Both of these species are white-rotting fungi("white-rotters") of the Basidiomycete family. Only in the case of P.obtusus has a partially pure pyranose-2-oxidase or glucose-2-oxidaseenzyme preparation, substantially free of cell material, been obtained.Janssen and Ruelius (1968), supra.

Glucose-2-oxidase activity, and by implication pyranose-2-oxidaseactivity, are not found in all fungal species, even in all white-rottingfungal species, and apparently not even in all species of any fungalgenus which has at least one species which has glucose- orpyranose-2-oxidase activity.

163 fungal strains, including the following 13 white-rotters:

(3) Laetiporus sulfureus M-4

(2) Laetiporus sulfureus F-4

(3) Phellinus gilvus

(4) Ganoderma applanatum

(5) Phellinus pini

(6) Pleurotus ostreatus

(7) Poria andersonii

(8) Stereum sp.

(9) Coriolus versicolor

(10) Ganoderma lucidum

(11) Lenzites betulinus

(12) Armillariella mellea

(13) Stereum hirssutum

were screened as follows for glucose-2-oxidase activity. The strainswere obtained from various sources, including nature and depositoriessuch as ATCC and NRRL. P. obtusus and O. mucida strains were notincluded. All strains were obtained and stored as slant cultures. The 13white-rotting strains were provided by Prof. Sara Fultz of StanfordUniversity, Palo Alto, California from her culture collection.

A culture of each of the 163 strains was grown on approximately 3 ml ofF4 agar, and another culture of each of the strains was grown onapproximately 3 ml of FA agar. F4 agar consists of 5.0 g tryptone, 3.0 gmalt extract, 10.0 g glucose, 3.0 g yeast extract and 0.2 g agarcombined with 1.0 l of distilled water (See Pansey, et al. Antimicrob.Agents Chemotherapy, 399 (1966). FA agar is prepared by combining 15.0 gglucose, 3.0 g yeast extract, 1 ml artificial sea water (from Aquariumsystems Inc., Eastlake, Ohio) and 0.2 g agar with 966 ml of distilledwater (See V. Lilly and H. Barnett, Physiology of the Fungi, McGraw-HillBook Co., Inc., New York, New York (1951)), then autoclaving the mixturefor 15 minutes at 15 psi pressure, and finally adding 100 microgramsthiamine and 5 microgram biotin via filter sterilization. Cultures onboth of the agars were grown and assayed in the same manner, as follows.

A culture was inoculated into the approximately 3 ml of agar, in acapped sterile vial, by picking the slant culture on which the strainwas received and stored, with a sterilized toothpick and thenasceptically touching the tip of the toothpick to the surface of theagar pad approximately at its center. Growth was continued for 2 to 4days at 25° C., as long as required for the organism to cover the agarpad. All of the above-listed 13 white-rotter strains were grown for 4days.

After the 1-4 day growth period, 5.0 ml of sterile 2% (w/v) glucosesolution, buffered to pH 6.0 with 35 mM potassium phosphate buffer, wasintroduced asceptically into the vial; and the vial was then shaken at250 rpm for 24 hours at 25° C.

After the shaking, 1.0 ml of fluid was removed from the vial andimmediately processed for testing for the presence of glucosone. Thefirst step in the processing was to add to the 1.0 ml of fluid 0.2 ml ofa solution of 2% (w/v) 2,4-dinitrophenylhydrazine in 30% perchloricacid. This solution was then analyzed by thin-layer chromatography (TLC)for the presence of the bis-2,4-dinitrophenylhydrazone of glucosone. TheTLC was carried out with a Selectasol® system from Schleicher andSchuell (Keene, New Hampshire) using 0.25 mm silica gel plates, 25%(v/v) xylene in ethyl acetate (a solution in which thebis-2,4-dinitrophenylhydrazone of glucosone is soluble and into which itcan be extracted from aqueous solution) as carrier, and a spray of 20%(v/v) 4.8M NaOH (in water) with ethanol to develop the plates. Thebis-2,4-dinitrophenylhydrazone of glucosone is purple on the developedplates.

The TLC assay could detect glucosone from a culture if as little as0.08% of the glucose in the culture system (after addition of the 2%glucose solution just prior to the 24-hour shaking) was present asglucosone at the time the 2,4-dinitrophenylhydrazine was added to the1.0 ml of culture removed for assay. A finding of detectable glucosonein the assay of a culture indicted the fungal strain in the culture hada glucose-2-oxidase activity of potential commercial value.

The same assay technique could have been used with xylose instead ofglucose to detect xylose-2-oxidase activity via the formation of thehydrazone of xylosone.

Of the 163 strains screened, only 2 were found to possessglucose-2-oxidase activity, both of which produced glucosone whencultured on either F4 or FA agar. The remaining 161 strains produced nodetectable glucosone when grown on either agar.

Both producing strains are from the 13 white-rotters listed above:Coriolus versicolor, now deposited in the CMCC under No. 0748 and in theNRRL under No. 15152, and Lenzites betulinus, now deposited in the CMCCunder No. 0749 and in the NRRL under No. 15593.

A culture of C. versicolor, deposited by us at NRRL, under No. 15152,and at CMCC under No. 0748, was grown out under culture conditionssimilar to those used to culture P. obtusus ATCC 26733, as detailed inExample I above. The cultured cells were plated on agar plates andselected for high producers of P2O, substantially as described inExample I, leading to cultures of substantially uniform, relatively highproducers. The best culture found produced P2O at a yield, expressed inInternational Units per liter of growth medium, after 6 days ofincubation, which was approximately 30% that produced by theabove-identified P. obtusus strain AU124.

This best culture of C. versicolor was assayed for PD activity by itsability to convert glucosone to a product having an absorption peak at265 nm. No PD activity was detected under a variety of assay conditionsat a variety of pH's.

A culture of L. betulinus, deposited at NRRL under No. 15593 and at CMCCunder No. 0749, was grown under conditions similar to those used toculture P. obtusus, ATCC 26733, as described in Example I. The L.betulinus culture was found to contain a 2 to 3-fold variability in theamount of P2O produced by cultures grown from individual cells, platedout from this initial culture, similar to what was observed for culturesderived from cultured cells of P. obtusus, ATCC 26733, and C. versicolorNRRL 15152. Cells from the L. betulinus culture were plated on agar, andselected for high producers of P2O, substantially as described inExample I, leading to cultures of substantially uniformly highproducers. The best culture found produced P2O at a yield, based onenzyme activity units per liter of growth medium, approximately 70% ofthat of the best culture of C. versicolor described above.

This best culture of L. betulinus, was assayed for PD activity as justdescribed, like C. versicolor. The results showed that L. betulinus alsoproduces substantially no detectable soluble PD.

EXAMPLE XI Preparation and Characterization of C. versicolor P2O

The high-producer culture of C. versicolor described in Example X wasgrown in submerged culture in 1.5 liters of yeast/malt extract medium at28° C. for 7 days. The resulting mycelial balls were collected byfiltration, washed, and then disrupted at 4° C. in 50 mM sodiumphosphate, pH 7.0, for 3 minutes using an Ultraturrax blender. Thelysate was cleared by centrifugation 25 minutes at 12,000 rpm. Toconcentrate and partially purify the enzyme, solid ammonium sulfate wasslowly added to 55% saturation, and the solution was allowed to standfor 90 minutes at 4° C., prior to collecting the precipitate. Theprecipitate was resuspended in 42 ml of 30 mM TRIS-HCl, pH 8.5, dialyzedagainst the same buffer, and centrifuged at 10,000 rpm for 15 minutes toremove a precipitate.

The enzyme was applied to a 2.6×40 cm DEAE column, washed, and elutedusing a 0-300 mM NaCl gradient. The P2O elutes at about 220 mM. Portionsof each fraction were assayed for P2O activity, by monitoring peroxidegeneration conventionally. Protein concentration was also determined bya conventional method. The fractions containing P2O activity were pooledand concentrated by ultrafiltration with an Amicon PM10 filter obtainedfrom Amicon Corp. Peak fractions were dialyzed into 30 mM sodium acetate(pH 5.5), adjusted to 1.5M NaCl and loaded onto a phenylsepharosecolumn. The P2O was eluted with a 0-1.5M NaCl gradient in 50 mM sodiumacetate, pH 5.5. The enzyme eluted at about 1.1M NaCl. Fractionscontaining the peak activity were pooled and concentrated to 0.5 ml byAmicon PM30 ultrafiltration and sized according to native molecularweight using a 1.5×95 cm Biogel P300 column. Elution was at 0.1ml/minute with 100 mM sodium phosphate, pH 7.0. Using known molecularweight markers in the column, the enzyme's apparent native molecularweight was calculated to be about 130,000 daltons. A single band havinga molecular weight of about 65,000 was observed when the enzyme wasanalyzed by SDS-PAGE. The enzyme thus appears to be a homodimer. Theenzyme was judged to be about 95% pure, based on the staining intensityof protein bands in SDS-PAGE.

Cofactor analysis indicated that each subunit of the enzyme contains aflavin moiety which appears to be covalently attached. The protein hasabsorption maxima at 276, 357 and 454 nm.

The relative activities of the enzyme on the substrates D-glucose,L-sorbose, D-xylose and delta-D-gluconolactone are 100: 74: 41: 6,respectively, at 1% (w/v) substrate concentration. The enzyme has a pHoptimum of about 5.0.

EXAMPLE XII Preparation and Characterization of P2O from L. betulinus

The high-producer culture of L. betulinus described in Example X wasgrown according to the conditions described in Example II. The cellswere homogenized and centrifuged to produce a supernatant, also inaccordance with the methods described in Example II. Polyethylene glycolwas added to the supernatant to a final concentration of 20%, and themixture was allowed to stand for 2 hours at 4° C., producing aprecipitate which was pelleted by centrifugation.

The resulting clear yellow supernatant was further purified using DEAEchromatography in 30 mM TRIS-HCl, pH 8.5, and eluted from the 5×16 cmcolumn using a 0-300 mM NaCl gradient. The protein eluted at about 75 mMNaCl. Fractions containing the peak of P2O activity were pooled andconcentrated to 0.5 ml by Amicon PM30 ultrafiltration, and thenfractionated, according to native molecular weight, using a 1.5×95 cmBiogel P300 column. The column was run at a flow rate of 0.1 ml perminute, using 100 mM sodium phosphate, pH 7.0. The protein eluted in afraction corresponding to an apparent native molecular weight of about300,000 daltons. Analysis of the purified protein by SDS-PAGE showed onemajor band corresponding to a molecular weight of about 65,000 daltons,and minor bands, presumably representing impurities. Based on themolecular weight data, the protein appears to be a homotetramer.

The enzyme contains a covalently attached flavin on each subunit, andhas absorption maxima at 275, 360, and 450 nm. The relative activitiesof the purified enzyme on the pyranose substrates D-glucose, L-sorbose,D-xylose and delta-D-gluconolactone are 100: 43: 43: 26, respectively,at 20 mM substrate concentration. The enzyme has a broad pH optimumbetween about 5 and 6.

EXAMPLE XIII Effect of H₂ O₂ on P2O Half-life

A P2O enzyme preparation was derived from P. obtusus AU124 in accordancewith the method detailed in Example II. A. niger catalase was obtainedfrom Fermco, Inc., and further purified by DEAE ion-exchangechromatography to remove glucose-1-oxidase activity present in thecommercially available enzyme, as in Example II. A phenol-formaldehydebead support, ES-762, was obtained from Diamond Shamrock (Foster City,CA).

Reagents containing the various activity ratios of P2O to catalase shownin Table I were prepared by sequential adsorption of first catalase andthen P2O to the support. The activity ratios of P2O to catalase in thefour supports is the theoretical steady-state level of H₂ O₂ in molesper liter expected in a P2O -reaction, assuming no inactivation ofeither enzyme.

                  TABLE I                                                         ______________________________________                                               P2O/Catalase    [H.sub.2 O.sub.2 ]                                                                    P2O Half Life                                  Reagent                                                                              (activity ratio in M)                                                                         (mM)    (Normalized)                                   ______________________________________                                        #1     no catalase     10      1                                              #2     0.67 × 10.sup.-3                                                                        2.0     8.0                                            #3     0.45 × 10.sup.-3                                                                        0.3     11.6                                           #4     0.07 × 10.sup.-3                                                                        0.04    28-60                                          ______________________________________                                    

Each of the supports was packed in a 7 mm×1.2M glass Pyrex columnreactor by pouring a slurry of the support over about 10 inches of 1 mmdiameter non-porous glass beads, and applying a low vacuum to the bottomto aid in settling the bed. When a packed column height of 25 inches wasreached, another 10 inches of glass beads was placed on the resin bedfollowed by approximately an inch of glass wool. After circulatingsterile water through the bed to wash out any excess reagents or coloredmaterial, the column was equilibrated with 25 mM citrate, pH 5.5.

The reaction was started up by introducing a 5% solution of glucose incitrate buffer through the column. The level of H₂ O₂ which eluted fromthe reactor column was monitored over a period of continuous columnoperation of about 12 days.

The reactor packed with reagent #1, containing only P2O, showed animmediate burst of H₂ O₂, to approximately 100 mM, followed by a steadydecline in H₂ O₂ concentration to about 10 mM by the third day ofcontinuous operation.

The reactor packed with reagent #2 showed a steady increase in H₂ O₂concentration from a level of about 0.3 to 3 mM, over the first four tofive days of reactor operation, indicating a slow inactivation ofcatalase, presumably by the H₂ O₂ being produced in the P2O reaction.From the maximum concentration of about 3 mM, the H₂ O₂ concentrationdeclined gradually to about 0.3 mM by the twelfth day of reactoroperation.

For both reagents #3 and #4, the reactor H₂ O₂ concentration startedfrom initial levels at or below about 0.3 mM, and remained substantiallyconstant over a several day reaction period, and then showed increasesof up to about 0.7 mM and 0.3 mM, respectively, by the twelfth day ofthe reaction. The increase in H₂ O₂ concentration suggests greater H₂ O₂inactivation of catalase than P2O. The H₂ O₂ concentrations in the fourreactors, measured after three days of column operation, are listed inthe third column in Table I. The measured concentrations of H₂ O₂ areconsistent with the theoretical H₂ O₂ concentrations calculated from theassociated P2O to catalase activity ratios.

For each of the four reagents, the half-life of the P2O enzyme wasdetermined by monitoring the rate of glucosone production in thecorresponding reaction during the 12-day course of the reaction.Glucosone levels produced by each reactor were measured using theabove-described triphenytetrazolium colorometric assay for glucosone.The half-life data are shown at the right-hand column in Table I. Theactual half-life values have been normalized to an arbitrary unit valueof 1 for the reagent #1 lacking catalase. The data show that thehalf-life of P2O is increased about 11-fold by employing a P2O tocatalase ratio which maintains the H₂ O₂ concentration below about3×10⁻⁴ M (reagent #3) and can be increased between about 28 and 60 foldby employing a P2O to catalase activity ratio which maintains the H₂ O₂concentration in the reactor below about 4×10⁻⁵ M.

EXAMPLE XIV Production of Substantially Pure Glucosone

A P2O enzyme preparation obtained from P. obtusus, strain AU124, wasprepared in accordance with Example II, and the P2O enzyme was purifiedsubstantially to homogeneity in accordance with Example III. The enzymewas used to convert glucose to glucosone under the following conditions:The purified P2O (0.46 units) and catalase were added to 500 microlitersof unbuffered medium, pH 5.0. Glucose was added to a final concentrationof about 5%. The reaction was carried out at room temperature, under 90%relative humidity, until all of the glucose substrate was consumed. ThepH of the solution remained constant at 5.0 throughout the reactionperiod without the addition of a base to the reaction mixture.

At the end of the reaction period, the reaction products were analyzedby HPLC, monitored at 192 nm, as described in Example IV. The absorptionprofile of the HPLC-fractionated products is shown in FIG. 5. Here it isseen that the reaction produces substantially pure glucosone. Theglucosone was judged to be greater than 99% pure, based on the relativeareas of the peaks associated with glucosone and reaction by-products.

The results of this study illustrate that where glucosone breakdown isprevented, according to the method of the invention, the pH of thereaction medium is substantially stable, thereby eliminating the need tocontrol reaction pH with a strong buffer and/or by the periodic additionof base to the reaction.

While preferred embodiments of the invention have been described withreference to the examples herein, it will be appreciated that variouschanges and modifications can be made without departing from the spiritand scope of the invention.

What is claimed is:
 1. A method of producing a substantially purepyranosone from the corresponding pyranose comprisingproviding apyranose-2-oxidase enzyme which is substantially free ofpyranosone-utilizing enzyme contaminants, including pyranosonedehydratase activity which have measurable activity at a selected pHbetween about 4.4 and 7.0, mixing the pyranose with the enzyme and an H₂O₂ -removing catalyst, and performing the enzyme reaction in thepresence of O₂ at the selected pH.
 2. The method of claim 1, whereinsaid providing includes eliminating substantially all pyranosonedehydratase activity at the selected pH.
 3. The method of claim 2,wherein said providing includes selecting, as a source of thepyranose-2-oxidase enzyme, a microorganism capable of converting glucoseto glucosone, but incapable of converting glucose or glucosone tocortalcerone.
 4. The method of claim 3, wherein the organism from whichthe pyranose-2-oxidase enzyme is derived is selected from the groupconsisting of C. versicolor and L. betulinus.
 5. The method of claim 4,wherein the pyranose-2-oxidase enzyme is purified substantially tohomogeneity.
 6. The method of claim 2, wherein said providing includesselecting as a source of the pyranose-2-oxidase enzyme, a microorganismcapable of converting glucose to glucosone and cortalcerone, and saideliminating includes purifying the pyranose-2-oxidase enzyme to removesubstantially all pyranosone dehydratase activity at pH 6.5.
 7. Themethod of claim 2, wherein said providing includes selecting as a sourceof the pyranose-2-oxidase enzyme, a microorganism capable of convertingglucose to glucosone and cortalcerone, and said eliminating includesheat-treating the pyranose-2-oxidase enzyme to inactivate pyranosonedehydratase preferentially.
 8. The method of claim 7, wherein theorganism from which the pyranose-2-oxidase is selected includes P.obtusus, and the pyranose-2-oxidase enzyme is prepared therefrom in thepresence of a protease inhibitor.
 9. The method of claim 8, wherein theprotease inhibitor includes phenylmethylsulfonyl fluoride.
 10. Themethod of claim 8, wherein said heat treating is performed at about pH5.0 for about 15 minutes at approximately 65° C.
 11. The method of claim2, wherein the the pyranose-2-oxidase enzyme provided is obtained fromP. obtusus, and contains substantial pyranosone dehydratase activity atpH 6.5, and said performing is carried out at pH 4.4, where the activityof the pyranosone dehydratase in the pyranose-2-oxidase enzyme is lessthan about 5% of its activity at its pH optimum of about pH 7.5.
 12. Themethod of claim 2, wherein the pyranose-2-oxidase enzyme is obtainedfrom a fungal culture, or a subculture thereof, produced by selectingfrom a mutagenized culture of a fungal organism, individual cellscapable of producing a level of pyranose-2-oxidase activity, as detectedin a broken-cell assay, that is several times that detected in brokencells of the unmutagenized organism.
 13. The method of claim 12, whereinthe selected organism includes P. obtusus, the mutagenic agent includesultraviolet light, and the culture produced has a detectablepyranose-2-oxidase activity which is about 5 times that of theunmutagenized cells.
 14. The method of claim 13, wherein themutagenized, high-producer P. obtusus culture has the characteristics ofP. obtusus strain AU124 cultures, deposited at NRRL under No.
 15595. 15.The method of claim 2, wherein said providing includes homogenizing P.obtusus mycelia in an aqueous medium, centrifuging the homogenate toform a supernatant, adding polyethylene glycol to the supernatant toprecipitate the pyranose-2-oxidase enzyme, and purifying the enzyme byion exchange chromatography to remove substantially all pyranosonedehydratase activity at pH 6.0 and below.
 16. The method of claim 2,wherein said providing includes homogenizing P. obtusus mycelia in abuffer containing a protease inhibitor, centrifuging the homogenate toform a supernatant, adding polyethylene glycol to the supernatant toprecipitate the pyranose-2-oxidase enzyme, resuspending the enzyme inbuffer, and heat-treating the suspended enzyme at approximately 65° C.for about 15 minutes until the enzyme solution contains substantially nopyranosone dehydratase activity at pH 6.0 or below.
 17. The method ofclaim 2, wherein said providing includes rupturing cells of a culture ofC. versicolor, centrifuging the ruptured cells to form a supernatant,precipitating the pyranose-2-oxidase enzyme by the addition of ammoniumsulfate, and purifying the enzyme, after resuspension in buffer, byion-exchange and molecular sieve chromatography.
 18. The method of claim2, wherein said providing includes disrupting mycelia of a culture of L.betulinus, centrifuging the ruptured cells to form a supernatant,precipitating the pyranose-2-oxidase enzyme by the addition ofpolyethylene glycol, and further purifying the enzyme by ion exchangechromatography.
 19. The method of claim 1, wherein the catalyst includescatalase which has been purified to remove substantially allglucose-utilizing enzymes, and the pyranose-2-oxidase/catalase activityratio is between about 10⁻⁵ and 10⁻³.
 20. The method of claim 19,wherein the catalase is obtained from Aspergillis niger, and is freed ofsubstantially all glucose-1-oxidase activity.
 21. The method of claim19, wherein the pyranose-2-oxidase and catalase enzymes areco-immobilized on a solid substrate, at an initialpyranose-2-oxidase/catalase activity ratio between about 5×10⁻⁵ and5×10⁻⁴.
 22. The method of claim 21, wherein the reaction is performed ina continuous-flow reactor.
 23. The method of claim 2, wherein the pH ofthe reaction is maintained at the selected pH by the rapid addition of aweak base.
 24. A method of producing substantially pure glucosone fromglucose comprisingproviding a pyranose-2-oxidase enzyme prepared byhomogenizing cells of a P. obtusus culture, centrifuging the homogenateto form a supernatant, precipitating the enzyme by the addition of anprotein-precipitating agent, and resuspending the enzyme in an aqueousmedium, where the glucose-to-glucosone conversion is to be carried outat a pH above about 4.4, treating the resuspended enzyme by one of thefollowing procedures: (a) further purifying the resuspended enzyme byion exchange chromatography to remove substantially all pyranosonedehydratase activity at pH 6.0; and (b) heat treating the resuspendedenzyme at approximately 65° C. for about 15 minutes; mixing glucose withthe resuspended enzyme and, in close physical association therewith,catalase, at a pyranose-2-oxidase/catalase activity ratio of betweenabout 5×10⁻⁵ and 5×10⁻⁴, and performing the enzyme reaction in thepresence of O₂, at a pH below about 6.0 for pyranose-2-oxidase enzymetreated according to procedures (a) and (b), and at a pH of about 4.4for untreated pyranose-2-oxidase enzyme.
 25. The method of claim 24,wherein the resistance of the pyranose-2-oxidase enzyme to heatinactivation, in accordance with procedure (b), is enhanced by adding aprotease inhibitor to the cell homogenate.
 26. The method of claim 24,wherein the heat treating according to procedure (b) is performed atabout pH 5.0.
 27. The method of claim 24, wherein the P. obtusus cultureis obtained from cells which have been mutagenized and selected fortheir ability to produce a high level of pyranose-2-oxidase.
 28. Themethod of claim 27, wherein the mutagenized, high-producer P. obtususculture has the identifying characteristics of a culture of P. obtususstrain AU124, deposited at NRRL under No.
 15595. 29. The method of claim24, wherein the catalase is obtained from Aspergillis niger, and ispurified to remove substantially all glucose-1-oxidase activity.
 30. Amethod of substantially eliminating acid-generating side reactions inthe enzymatic conversion of a glucose to glucosone by pyranose-2-oxidasein the presence of oxygen and a hydrogen peroxide-destroying catalyst,thus to stabilize the the pH of the enzymatic conversion reaction mediumat an initially selected reaction pH between about 4.4 and 6.5, saidmethod comprising eliminating from the reaction medium substantially allpyranosone-utilizing enzyme activity including pyranosone dehydrataseactivity at the initially selected pH.
 31. The method of claim 30,wherein the reaction medium is adjusted initially to pH 5.0.
 32. Themethod of claim 30, wherein said eliminating includes using apyranose-2-oxidase which is substantially free of all pyranosedehydratase activity at pH 6.0.
 33. The method of claim 30, wherein saideliminating includes using a pyranose-2-oxidase enzyme which has beenheat-treated to inactivate substantially all pyranosone dehydrataseactivity at pH 6.0.
 34. The method of claim 30, wherein said eliminatingincludes using a pyranose-2-oxidase enzyme which has been partiallypurified to remove substantially all pyranosone dehydratase activity atpH 4.4.
 35. The method of claim 30, wherein said eliminating includesusing a pyranose-2-oxidase enzyme which is obtained from a fungalorganism capable of converting glucose to glucosone but incapable ofconverting glucose or glucosone to cortalcerone.
 36. The method of claim35, wherein the organism is selected from one of the group consisting ofC. versicolor and L. betulinus.
 37. A method of producing an enzymereagent capable of converting glucose to substantially pure glucosone,at a selected pH between about 4.4 and 7.0, said methodcomprisingproviding a pyranose-2-oxidase enzyme which is substantiallyfree of glucosone-utilizing enzyme contaminants including pyranosonedehydratase which have measurable activity at the selected pH at whichthe glucose-to-glucosone conversion is to be carried out, providingcatalase which has been substantially purified of glucose-utilizingenzyme contaminants, and attaching the pyranose-2-oxidase and catalaseenzymes to a solid support in a pyranose-2-oxidase to catalase activityratio of between about 10⁻⁵ to 10⁻³.
 38. The method of claim 37, whereinsaid providing includes obtaining a partially purified glucose-2-oxidaseenzyme from P. obtusus and, where the glucose-to-glucosone conversion isto be carried out at a pH of above about pH 4.4, further treating theglucose-2-oxidase enzyme to eliminate substantially all pyranosonedehydratase activity at a pH of about 6 and below by one of thefollowing procedures:(a) fractionating the partially purified enzyme byion exchange chromatography to remove pyranosone dehydratase; and (b)heat treating the partially purified enzyme at approximately 65° C. forabout 15 minutes, to inactivate substantially all pyranosone dehydrataseactivity at pH 6.0.
 39. The method of claim 38, which further includesmutagenizing cells of a P. obtusus culture, selecting mutant cells whichproduce a level of pyranose-2-oxidase which is several times thatproduced by unmutagenized cells, and preparing the pyranose-2-oxidaseenzyme from a culture of the selected cells.
 40. The method of claim 39,wherein the culture of selected P. obtusus cells has the characteristicsof a culture of P. obtusus strain AU124, deposited at NRRL under No.15595.
 41. The method of claim 37, wherein said providing includesobtaining the pyranose-2-oxidase enzyme from a fungal organism capableof converting glucose to glucosone, but incapable of converting eitherglucose or glucosone to cortalcerone.
 42. The method of claim 41,wherein the organism is selected from one of the group consisting of C.versicolor and L. betulinus.
 43. The method of claim 37, wherein thecatalase is isolated from Aspergillis niger, and is purified to removesubstantially all glucose-1-oxidase activity.
 44. An enzyme reagent forconverting glucose to substantially pure glucosone, at a selected pHbetween about 4.4 and 7.0, comprisinga solid support, and molecules ofpyranose-2-oxidase and catalase attached to the surface of the supportat a pyranose-2-oxidase/catalase activity ratio of between about 10⁻⁵ to10⁻³, said pyranose-2-oxidase being produced by homogenizing cells of aP. obtusus culture to obtain a homogenate, centrifuging the homogenateto form a supernatant, precipitating the glucose-2-oxidase by theaddition of a protein-precipitating agent, resuspending the precipitatedenzyme in a aqueous medium, and where the reagent is to be used in areaction which is carried out above about pH 4.4, further treating theresuspended enzyme to eliminate substantially all glucosone-utilizingenzyme activity above about pH 6.0 by one of the following procedures:(a) further purifying the pyranose-2-oxidase to remove substantially allpyranosone dehydratase activity at pH 6.0; and (b) heat treating theresuspended enzyme at approximately 65° C. for about 15 minutes, saidcatalase being freed of substantially all glucose-utilizing activity.45. The reagent of claim 44, wherein the activity ratio ofglucose-2-oxidase to catalase is less than about 5×10⁻⁴.